Role of TGFbeta in Microtubule Dynamics

描述(由申请人提供)：在响应生长因子(GFS)的信号转导过程中，通过内吞途径的区隔和功能专门化可以实现更高级别的调节。相反，GF信号复合体细胞内(IC)运输的障碍会导致信号分子在错误的间隔或错误的时间内错误定位或聚集，从而导致包括癌症在内的各种疾病状态。此外，运动蛋白轻链(LCS)在调节GF信号转导过程中募集其膜上货物进行IC转运的特异性也日益明确。在这些新的信号机制的基础上，需要对来自内体亚室的信号的调节、关键成分、时间和功能结果进行额外的研究，以支持肿瘤药物的开发。 我们上一个项目期的数据表明，km23 dynein LCS被转化生长因子？受体(T？RS)是Smad转运所必需的，在核转位之前是必需的。因此，km23似乎是一种“运动受体”，可以招募转化生长因子？动力蛋白马达的信号复合体，用于沿着微管(MT)向细胞核进行IC运输货物(即T？RS、SMADS)。此外，我们还鉴定了km23/LC7/Rob1/DYNLRB家族的另一个相关成员，称为km23-2。虽然这个异构体似乎具有一些类似于km23-1的特征，但km23-2似乎更特异地调控Smad3，而不是Km23-1对Smad2的调控。在新的应用中将探索Smads2/3与km23-1/2马达LC相关的不同调控和贩运的机制，包括km23如何影响下游的转化生长因子？回应。在上一个项目期间，我们还证明了蛋白激酶A(PKA)直接磷酸化km23，并且是km23与动力蛋白中间链(DIC)结合所必需的。这种PKA在转化生长因子中磷酸化km23的机制是什么？还将审查信令和Smad2/3贩运。 其他研究涉及这些贩运事件在已知表达改变形式的km23-1的人卵巢癌细胞(HOCC)中如何改变的调查。作为最后一个项目的一部分，我们在42%的卵巢癌患者组织中发现了km23-1改变，在正常组织中没有检测到km23改变。卵巢癌中如此高的转移率表明km23可能在卵巢癌的TGF2耐药或肿瘤进展中起重要作用。在新的应用中，我们将研究敲除km23或在关键磷酸化位点突变它对Smad运输、Smad特异性转化生长因子？以及体外人类癌细胞的恶性表型，以及体内肿瘤进展的影响。

DESCRIPTION (provided by applicant): A higher order of regulation can be achieved by compartmentalization and functional specialization of the endocytic pathway during signal transduction in response to growth factors (GFs). In contrast, impairment of intracellular (IC) trafficking of GF signaling complexes can result in mis-localization or accumulation of signaling molecules in the wrong compartment, or at the wrong time, giving rise to various disease states, including cancer. In addition, the specificity of motor protein light chains (LCs) in recruiting their membranous cargo for IC transport during regulation of GF signaling is becoming increasingly clear. Additional investigation of the regulation, key components, timing, and functional outcome of signaling from endosomal subcompartments is required to enable oncology drug development, based upon these newer signaling regimes. Our data from the last project period indicated that km23 dynein LCs are activated by TGF? receptors (T?Rs) and are required for Smad trafficking, prior to nuclear translocation. Thus, km23 appears to function as a "motor receptor" to recruit TGF? signaling complexes to the dynein motor for IC transport of cargo (ie, T?Rs, Smads) along microtubules (MTs) toward the nucleus. In addition, we have identified another related member of the km23/LC7/robl/DYNLRB family, termed km23-2. While this isoform appears to have some characteristics similar to km23-1, km23-2 appears to more specifically regulate Smad3, in contrast to Smad2 regulation by km23-1. The mechanisms underlying the differential regulation and trafficking of Smads2/3 in relation to the km23-1/2 motor LCs will be explored in the new application, including how km23 impacts downstream TGF? responses. During the last project period, we have also shown that protein kinase A (PKA) directly phosphorylates km23 and is required for km23 binding to the dynein intermediate chain (DIC). The mechanisms underlying this PKA phosphorylation of km23 in TGF? signaling and Smad2/3 trafficking will also be examined. Additional studies relate to the investigation of how these trafficking events are altered in human ovarian cancer cells (HOCCs) known to express altered forms of km23-1. As part of the last project period, we identified km23-1 alterations in 42% of ovarian cancer patient tissues, with no km23 alterations detectable in normal tissues. Such a high alteration rate in ovarian cancer suggests that km23 may play an important role in either TGF2 resistance or tumor progression of this disease. In the new application, we will examine the effects of knocking down km23, or mutating it at key phosphorylation sites, on Smad trafficking, Smad-specific TGF? responses, and the malignant phenotype of the human cancer cells in vitro, as well as on tumor progression in vivo.